The overall purpose of Dr. O'Donnell's Gene Expression for Functional Genomics in Human Populations research program during FY 2015 is to define the functional genomics of high interest genes using targeted and genomewide gene expression analysis. With these functional experiments, wee seek to understand the biological underpinnings of the genetic/genomic epidemiology of subclinical and clinical atherosclerotic cardiovascular disease (CVD) and its risk factors, including hemostatic and thrombotic risk. The longer term goal is to translate these results to prediction, prevention and personalization of CVD medicine. The major projects have emanated from the SNP Health Association Resource (SHARe), the Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) Consortium, and collaborative DNA and RNA sequencing projects. Dr. O'Donnell is the Associate Director of the NHLBI's Framingham Heart Study (FHS), Scientific Director and Steering Committee Chair of the NHLBI's Framingham Heart Study (FHS) SHARe Program, co-founder and Steering Committee Co-chair of the CHARGE Consortium, and Co-Director of the HeartGO Consortium of the NHLBI GO Exome Sequencing Project (ESP). Research Subjects: The research subjects for RNA Sequence Analysis consist primarily of 180 participants from the Framingham Heart Study FHS original Offspring cohort (N=60 with prior myocardial infarction, N=60 with high levels of CAC, and n=60 free of coronary disease or CAC). Additionally, we are conducitng gene expression analysis of lymphocyte- and platelet-derived RNA using rtPCR and of whole genome RNA profiling (Affymetrix Exon Array) in the FHS Offspring and Gen3 cohorts. Additional aortic arterial endothelial cells (n=15 individual donors) and coronary artery endothelial cells (n=12 individual donors) are available from normal humans (purchased from a vendor) and are used for study. Phenotyping: Phenotyping of FHS participants consisted of: (a) risk factor measures from usual clinical exams (lipids, blood pressure, anthropometric and physical examination); (b) biomarkers from peripheral blood (eg, C-reactive protein, fibrinogen, von Willebrand factor); (c) imaging measures of subclinical atherosclerosis (coronary and abdominal and thoracic aortic atherosclerosis by multidetector CT imaging (MDCT) in 3500 Offspring and Generation 3 subjects; carotid intimal medial thickness (CIMT) and carotid plaque by B-mode ultrasonography in 3800 Offspring: aortic plaque, LV mass by cardiovascular magnetic resonance imaging (CMRI) in 1800); (d) clinical CVD outcomes (myocardial infarction; coronary heart disease; CVD) adjudicated by a physician endpoint validation committee; (e) gene expression of lymphocyte- and platelet-derived RNA using rtPCR, whole genome RNA profiling (Affymetrix Exon Array), and next-generation RNA sequencing. No phenotypes are available for the n=15 arterial endothelial cells and n=12 coronary artery endothelial cells. Genotyping and resequencing: Genotyping derived from two dense genomewide SNP scans, a 100K SNP scan (Affymetrix platform) in 1400 FHS Offspring and original cohort subjects and a 550K SNP scan (Affymetrix platform, 250K Nsp and 250K Sty and 50K gene-focussed MIP) in 9,400 FHS subjects from all three generations. Imputation of the 550K SNPs was conducted to 2.4 million HapMap SNPs using MACH and to 40 million SNPs from the 1000 Genomes Project. Additional SNP genotyping is being conducted using various platforms including 250K functional exon variants on the exome chip (Illumina). Exome chip genotyping has been conducted (Affymetrix) in the n=15 human endothelial cell samples. Research Accomplishments for Projects Directed by Dr. O'Donnell in FY 2015: 1. Characteristics of Common Genetic Variants Controlling Transcript Isoform Variation in the Framingham Heart Study: Alternative splicing (AS) affects 80% of human pre-mRNAs. We performed genome-wide screening to identify SNPs from 1000G imputed SNP set controlling splicing in whole blood collected from the Framingham Heart Study. Many strong common cis-acting regulatory variants affect the splicing patterns of genes in a large population, including SNPs known to be associated with disease traits from prior genomewide association studies. A manuscript describing these findings is published. 2. RNA Transcriptome Sequencing of Myocardial Cases, High Levels of Coronary Artery Calcium, and Controls: This Myocardial Infarction Transcriptome Sequencing project aims to use RNA sequencing to identify differentially expressed transcripts, possibly due to genetic alterations, in individuals who undergo early myocardial infarction (MI) as contrasted with age-sex matched individuals who do not. The resulting transcriptome data from this project will include gene expression profiles of cases of MI or high CAC and controls (known and novel splicing variants, long noncoding RNAs) as well as allelic expression imbalance data in heterozygotes. Sequencing is completed and a manuscript in preparation. 3. Characterizing the Aorta Vascular Endothelial Cell Transcriptome with RNA Sequencing: We have completed a deep RNA sequencing experiment to quantitate the expression level of mRNAs and large intergenic noncoding RNAs (lincRNAs) in aorta vascular endothelial cells. Detailed information regarding risk alleles is available from genotyping of the endothelial cell lines (Affymetrix). Initial analysis confirms the presence of a unique transcript profile and unique lincRNAs for aortic vascular endothelial cells. Manuscript is in preparation. 4. Creation of induced pluripotent stem cells and groundwork for genome editing experiments: Most of the n=14 Aorta Vascular Endothelial Cell lines are being reprogrammed into induced pluripotent stem cells in the NHLBI iPS Core Laboratory. Protocol development is completed for differentiating these iPS cell lines into aortic vascular endothelial cells and protocol development underway to differentiate iPS cells to aortic endothelial cells and to conduct genome editing using the CRISP-Cas system. 5. Collaboration with FHS Next Generation induced pluripotent stem cell project: RNA Sequencing is underway in iPS cells as well as cells differentiated from these iPS cells into hepatocyte and adipocyte lineages.